1. Field of the Invention
The invention generally relates to bacterial delivery systems that promote improved transgene expression in eukaryotic cells by inhibiting the innate type I interferon response. In particular, the invention provides recombinant bacterial delivery systems that deliver to eukaryotic cells: i) transgenes and ii) suppressors of the eukaryotic Type I interferon response.
2. Background of the Invention
Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of targeted delivery of macromolecules to mammalian cells and tissues in a single oral, intranasal or inhalational dose, thereby stimulating both systemic and mucosal immune responses. The great potential of bacteria-mediated transfer of plasmid DNA encoding vaccine antigens and/or therapeutic molecules has been demonstrated in experimental animal models of infectious diseases, tumors and gene deficiencies.
Unfortunately, bacterial vectored discharge of passenger RNA/DNA and other molecules for the expression of foreign proteins or inhibitory RNAs in mammalian cells results in a type I interferon (IFN) response. A central component of the host's surveillance system for invading pathogens is an evolutionarily conserved family of pathogen recognition receptors (PRR) which bind patterned microbial/viral ligands ranging from cell wall components to nucleic acids. PRR signaling results in the activation of transcription factors such as Nuclear Factor-B (NF-B) and interferon regulatory factor 3 (IRF-3), which provide the inflammatory context for the rapid activation of host defenses. The NF-B pathway controls the expression of proinflammatory cytokines such as IL-1 and tumor necrosis factor-α, whereas the IRF-3 pathway leads to the production of type I inteferons (IFN-α and IFN-β). This initially produced “first wave” IFN triggers expression of a related factor, IRF-7, which is normally present in most cells at very low concentrations (Sato M et al., Immunity, 13(4)539-548; 2000). IRF-3 most likely cooperates with IRF-7 and is responsible for a positive feed back loop that initiates the synthesis of several IFN-α subtypes as the “second wave” IFNs (Marie et al., EMBO J 17(22), 6660-6669; 1998 and Sato M et al., FEBS Lett 441(1)106-110; 1998). Type I IFNs activate several hundred IFN stimulated genes by autocrine and paracrine signaling (ISGs) (de Veer et al., J Leukocyte Biol 69(6) 912-920, 2001; Der et al., Proc. Natl. Acad. Sci. USA 95(26) 15623-15628; 1998), some of which code for antiviral proteins. To date, three IFN stimulated pathways have been firmly established. These include protein kinase R (PKR) (Williams Oncogene 18(45) 6112-6120; 1999), the 2′-5′ oligoadenylate-synthetase (2′-5′ OAS) (Silverman, J Interferon Res 14(3) 101-104; 1994) and the Mx proteins (Haller and Kochs Traffic 3(10) 710-714; 2002). This type I IFN response limits the expression of foreign genes or inhibitory RNAs by means of PKR and 2′-5′ OAS. Activated PKR blocks translation by phosphorylating the a subunit of eukaryotic initiation factor eIF2. On the other hand, 2-5 A synthetases produce short, 2′-5′ OAS associated oligoadenylates which activate RNase L, a single-stranded specific endoribonuclease that digests mRNA and ribosomal RNA. The importance of the Mx protein in host survival following infection with certain RNA viruses has been amply demonstrated (Hefti et al., J Virology 73(8) 6984-6991; 1999) but the exact mode of action is still unknown. This type I IFN response thus limits the expression of foreign nucleic acids by mechanisms which reduce RNA production and stability and also inhibits translation of message from passenger nucleic acids delivered by a bacterial vector.
Various components of bacterial vectors elicit the IFN response in host cells. The bacterium itself can trigger an IFN response through Toll-like receptors. Double stranded RNA produced by passenger nucleic acids during transcription not only induces type I IFNs but also directly activates PKR and 2′-5′ OAS. Plasmid DNA, upon its delivery into the cytoplasm of mammalian cells, often contains cryptic promoters that generate anti-sense RNA which anneals with mRNA to form dsRNA. All these components of bacterial vectors thus diminish the efficacy of bacterial vectors as biomedical tools.
U.S. Pat. No. 6,525,029 (Falck-Perersen et al., Feb. 25, 2003) describes methods of inhibiting an immune response to a recombinant vector such as an adenoviral vector. However, this technology is directed toward preventing humoral (e.g. antibody) responses to long-term expression of genes encoded by a vector and clearance of the vector by the immune system, and does not address prevention of a type I IFN response to a bacterial vector or its passenger nucleic acids.
The prior art has thus-far failed to provide bacterial vectors that eliminate or attenuate the type I IFN response of host cells.